The family Pasteurellaceae encompasses several significant pathogens that infect a wide variety of animals. In addition to P. multocida, prominent members of the family include Pasteurella (Mannheimia) haemolytica, Actinobacillus pleuropneumoniae and Haemophilus somnus. P. multocida is a gram-negative, nonmotile coccobacillus which is found in the normal flora of many wild and domestic animals and is known to cause disease in numerous animal species worldwide [Biberstein, In M. Kilian, W. Frederickson, and E. L. Biberstein (ed.), Haemophilus, Pasteurella, and Actinobacillus. Academic Press, London, p. 61-73 (1981)]. The disease manifestations following infection include septicemias, bronchopneumonias, rhinitis, and wound infections [Reviewed in Shewen, et al., In C. L. Gyles and C. O. Thoen (ed.), Pathogenesis of Bacterial Infections in Animals. Iowa State University Press, Ames, p. 216-225 (1993), incorporated herein by reference].
Infection by P. multocida generally results from invasion during periods of stress, but transmission may also occur by aerosol or contact exposure, or via flea and tick vectors. In fowl, P. multocida infection gives rise to acute to peracute septicemia, particularly prevalent in domestic turkeys and wild waterfowl under stress conditions associated with overcrowding, laying, molting, or severe climatic change. In cattle, a similar hemorrhagic septicemia follows infection and manifests conditions including high fever and depression, generally followed by quick death. Transmission is most likely through aerosol contact, but infection can also arise during periods of significant climatic change. In rabbits, infection gives rise to recurring purulent rhinitis, generally followed by conjunctivitis, otitis media, sinusitis, subcutaneous abscesses, and chronic bronchopneumonia. In severe infections, rabbit mortality arises from acute fibrinous bronchopneumonia, septicemia, or endotoxemia. Disease states normally arise during periods of stress. In pigs, common P. multocida disease states include atrophic rhinitis and bacterial pneumonia. Similar pneumonia conditions are also detected in dogs, cats, goats, and sheep. P. multocida is commonly detected in oral flora of many animals and is therefore a common contaminant in bite and scratch wounds.
P. multocida strains are normally designated by capsular serogroup and somatic serotype. Five capsular serogroups (A, B, D, E, and F) and 16 somatic serotypes are distinguished by expression of characteristic heat-stable antigens. Most strains are host specific and rarely infect more than one or two animals. The existence of different serotypes presents a problem for vaccination because traditional killed whole cell bacteria normally provide only serotype-specific protection. However, it has been demonstrated that natural infection with one serotype can lead to immunological protection against multiple serotypes [Shewen, et al., In C. L. Gyles and C. O. Thoen (Ed.), Pathogenesis of Bacterial Infections in Animals. Iowa State University Press, Ames, p. 216-225 (1993)] and cross protection can also be stimulated by using inactivated bacteria grown in vivo [Rimler, et al., Am J. Vet. Res. 42:2117-2121 (1981)]. One live spontaneous mutant P. multocida strain has been utilized as a vaccine and has been shown to stimulate a strong immune response [Davis, Poultry Digest. 20:430-434 (1987), Schlink, et al., Avian Dis. 31(1):13-21 (1987)]. This attenuated strain, however, has been shown to revert to a virulent state or cause mortality if the vaccine recipient is stressed [Davis, Poultry Digest. 20:430-434 (1987), Schlink, et al., Avian Dis. 31(1):13-21 (1987)].
Another member of the Pasteurella family, A. pleuropneumoniae exhibits strict host specificity for swine and is the causative agent of highly contagious porcine pleuropneumonia. Infection normally arises in intensive breeding conditions, and is believed to occur by a direct mode of transmission. The disease is often fatal and, as a result, leads to severe economic loss in the swine producing industry. A. pleuropneumoniae infection may be chronic or acute, and infection is characterized by a hemorrhagic, necrotic bronchopneumonia with accompanying fibrinous pleuritis. To date, bacterial virulence has been attributed to structural proteins, including serotype-specific capsular polysaccharides, lipopolysaccharides, and surface proteins, as well as extracellular cytolytic toxins. Despite purification and, in some instances cloning, of these virulence factors, the exact role of these virulence factors in A. pleuropneumoniae infection is poorly understood.
Twelve serotypes of A. pleuropneumoniae have been identified based on antigenic differences in capsular polysaccharides and production of extracellular toxins. Serotypes 1, 5, and 7 are most relevant to A. pleuropneumoniae infection in the United States, while serotypes 1, 2, 5, 7, and 9 are predominant in Europe. There are at least three significant extracellular toxins of A. pleuropneumoniae that are members of the haemolysin family and are referred to as RTX toxins. RTX toxins are produced by many Grarn negative bacteria, including E. coli, Proteus vulgarisa, and Pasteurella haemolytica, and the proteins generally share structural and functional characteristics. Toxins from the various serotypes differ, however, in host specificity, target cells, and biological activities.
The major A. pleuropneumoniae RTX toxins include ApxI, ApxII, and ApxIII. ApxI and ApxII have haemolytic activity, with ApxI being more potent. ApxIII shows no haemolytic activity, but is cytotoxic for alveolar macrophages and neutrophils. Most A. pleuropneumoniae serotypes produce two of these three toxins. For example, serotypes 1, 5, 9, and 11 express ApxI and ApxII, and serotypes 2, 3, 4, 6, and 8 express ApxII and ApxIII. Serotype 10, however, produces only ApxI, and serotypes 7 and 12 express only ApxII. Those A. pleuropneumoniae serotypes that produce both ApxI and ApxII are the most virulent strains of the bacteria.
The Apx toxins were demonstrated to be virulence factors in murine models and swine infection using randomly mutated wild type bacteria [Tascon, et al., Mol. Microbiol. 14:207-216 (1994)]. Other A. pleuropneumoniae mutants have also been generated with targeted mutagenesis to inactivate the gene encoding the AopA outer membrane virulence protein [Mulks and Buysee, Gene 165:61-66 (1995)].
At least eleven serotypes (1, 2, 5-9, 12-14 and 16) have been demonstrated within Mannheimia [Pasteurella]haemolytica [Angen, et al., Vet Microbiol 65(4):283-90 (1999)], a Pasteurellaceae species which is responsible for serious outbreaks of acute pneumonia in neonatal, weaned, growing and adult lambs, calves, and goats [Ackemmann, et al., Microbes Infect 2(9):1.079-88 (2000)]. Transportation, viral infections, overcrowding, and other stressful conditions predispose animals to M. haemolytica infection [Ackermann, et al., supra.] The leukotoxin (Lkt) of M. haemolytica is believed to play a significant role in pathogenesis, causing cell lysis and apoptosis that lead to the lung pathology characteristic of bovine shipping fever [Highlander, et al., Infect Immun 68(7):3916-22 (2000)] as well as lung injury in bovine pneumonic pasteurellosis [Jeyaseelan, et al., Microb Pathog 30(2):59-69 (2001)]. Lkt is a pore-forming exotoxin that has the unique property of inducing cytolysis only in ruminant leukocytes and platelets [Jeyaseelan, et al., (2001), supra.]. Cytolysis of many cell types is mediated by arachidonic acid (AA) and its generation by phospholipases is regulated by G-protein-coupled receptors [Jeyaseelan, et al., (2001) supra] Recent studies indicate that M. haemolytica Lkt binds to bovine CD18, the common subunit of all beta2 integrins [Jeyaseelan, et al., Infect Immun 68(1):72-9 (2000)]. It has also been shown that LFA-1 is a Lkt receptor, Lkt binding to LFA-1 is not target cell specific, Lkt binding to bovine LFA-1 correlates with calcium elevation and cytolysis, and bovine LFA-1 expression correlates with the magnitude of Lkt-induced target cell cytolysis [Jeyaseelan, et al., Infect Immun 68(1):72-9 (2000)].
In attempts to produce vaccine compositions, traditional killed whole cell bacteria have provided only serotype-specific protection [MacInnes and Smart, supra], however, it has been demonstrated that natural infection with a highly virulent serotype can stimulate strong protective immunity against multiple serotypes [Nielsen, Nord Vet Med. 31:407-13 (1979), Nielsen, Nord Vet Med. 36:221-234 (1984), Nielsen, Can J Vet Res. 29:580-582 (1988), Nielsen, ACTA Vet Scand. 15:80-89 (1994)]. One defined live-attenuated vaccine strain producing an inactive form of the ApxII toxin has shown promise for cross protection in swine [Prideaux, et al., Infection & Immunity 67:1962-1966 (1999)], while other undefined live-attenuated mutants have also shown promise [Inzana, et al., Infect Immun. 61:1682-6, (1993), Paltineanu, et al., In International Pig Veterinary Society, 1992, p. 214, Utrera, et al., In International Pig Veterinary Society, 1992, p. 213].
Because of the problems associated with vaccine formulations comprising bacterial strains with undefined, spontaneous mutations, there exists a need in the art for rational construction of live attenuated bacterial strains for use in vaccines that will safely stimulate protective immunity against homologous and heterologous Pasteurellaceae serotypes. There further exists a need to identify attenuated bacterial strains and genes required for bacterial virulence, thereby facilitating development of methods to identify anti-bacterial agents.